Anti-Mullerian Inhibiting Substance Type II Receptor (MISIIR) Immunoconjugates to Detect and Treat Cancer

ABSTRACT

Compositions and methods for detecting and treating cancers expressing Mullerian inhibiting substance Type II receptor (MISIIR) are provided.

This application is a continuation-in-part of PCT/US2004/022068, filedJul. 8, 2004, which claims priority to U.S. Provisional Application60/485,622 filed Jul. 8, 2003. The entire disclosure of each of theseapplications is incorporated by reference herein.

Pursuant to 35 U.S.C. Section 202(c), it is acknowledged that the UnitedStates Government has certain rights in the invention described herein,which was made in part with funds from United States National CancerInstitute, Grant No. 2P50 CA83638.

FIELD OF THE INVENTION

The present invention relates to the fields of immunology and cancertreatment. Specifically, compositions and methods for detecting andtreating cancers expressing Mullerian inhibiting substance Type IIreceptor (MISIIR) are disclosed.

BACKGROUND OF THE INVENTION

Several publications and patent documents are cited throughout thespecification in order to describe the state of the art to which thisinvention pertains. Each of these citations is incorporated herein byreference as though set forth in full.

The precise targeting specificity of antibody molecules makes themattractive vehicles for tumor imaging and therapy agents. Employing anantibody with a high affinity for an antigen that is uniquely orpredominantly expressed on cancer cells can lead to a greaterspecificity of tumor retention, thereby increasing the likelihood ofsuccessful treatment or detection. While the initial clinical trials ofantibody-based tumor targeting revealed a number of hurdles (e.g., theimmunogenicity of the murine monoclonal antibodies, the poor tumorpenetration of immunoconjugates and a lack of tumor specificity of theantigenic targets), these have largely been addressed over the pastdecade. Anti-tumor antibodies are now developed using humanimmunoglobulin genes, resulting in proteins that are rarely seen asforeign. The degree of tumor penetration and the rate of systemicelimination of toxic immunoconjugates have been improved through thedevelopment of novel, minimal antibody-based structures (e.g.,monovalent and divalent single-chain Fv [scFv] molecules). Inpreclinical models, this has simultaneously increased the fraction ofthe tumor that is treated and reduced the exposure of normal tissues(e.g., bone marrow) to the immunoconjugate (Adams, G. P. (1998) In Vivo,12:11-22). Additionally, a second generation of tumor-associated ortumor-specific antigenic targets combines a predominantly tumor-specificexpression pattern with an antibody-triggered alteration inintracellular signaling. The targeting of cytotoxic agents, such asradioisotopes or drugs, to such an antigen can lead to an additive orsynergistic anti-tumor effect.

Monoclonal antibody (MAb)-based immunotherapy has shown notable promisein the treatment of hematologic malignancies (Kaminski, M. S., et al.(1993) N. Engl. J. Med., 329:459-465; Press, O. W., et al. (1993) N.Engl. J. Med., 329:1219-1224). Despite some constraints imposed by tumorphysiology (Jain, R. K., et al. (1987) 47:3039-3051), two of the fourmembers of the HER/EGF receptor family have proven to be useful targetsfor antibody-based therapy of cancer. MAbs specific for HER2/neu(Harweth, I. M., et al. (1993) Br. J. Cancer, 68:1140-1145) and EGFR(Fan, Z., et al. (1993) 53:4322-4328) can inhibit the growth of humantumors that overexpress their respective target antigens inimmunodeficient mice. Treatment with a humanized form of theanti-HER2/neu 4D5 MAb (HERCEPTIN™) leads to clinical responses alone andin combination with chemotherapy agents in clinical trials and islicensed for use in breast cancer (Baselga, J., et al. (1996) J. Clin.Oncol., 14:737-744; Baselga, J. (2001) J. of Cancer, 37 Suppl:S18-24). Anumber of clinical trials also have focused on the efficacy ofantibodies that target therapeutic radioisotopes to tumors(radioimmunotherapy or RAIT). Of note, the U.S. Food and DrugAdministration (F.D.A.) has approved commercial RAIT drugs ZEVALIN™, aconjugate of an anti-CD20 monoclonal antibody and the beta-emittingradioisotope Yttrium-90 [⁹⁰Y] for the treatment of non-Hodgkin'slymphoma, and BEXXAR®, iodine-131 [¹³¹I] Tositumomab for the treatmentof non-Hodgkin's lymphoma.

Radiolabeled MAbs have also been used for the radioimmunodetection(RAID) of a number of types of tumors including pancreatic cancer (Gold,D., et al. (2001) Crit. Rev. Oncol.-Hemat., 39:147-154), non small celllung cancer (Schillaci, O., et al. (2001) Anticancer Res.,221:3571-3574), ovarian cancer (Kalofonos, H. P., et al. (1999) ActaOncol., 38:629-634), colorectal carcinoma (Wong, J., et al. (1997) J. ofNuc. Med., 38:1951-1959; Willkomm, P., at al. (2000) J. of Nuc. Med.,41:1657-1663), and prostate cancer (Fang, D. X., et al. (2000) Tech.Urology, 6:146-150). These studies report the ability to detect lesionsthat are detectable by other methodologies (e.g., computerizedtomography (CT) and magnetic resonance imaging; MRI). Immunodetectioncan be improved upon, however, by employing smaller, engineeredantibody-based molecules such as scFv (Begent, R. H., et al. (1996) Nat.Med., 2:979-984). When isotopes with relatively short half-lives are tobe used, molecules like the small, divalent diabody should exhibit thegreatest degree of specific tumor localization in a setting of lownormal organ background (Williams, L. E., et al. (2001) Cancer Biother.And Radiopharm., 16:25-35). Furthermore, RAID studies can be effectivelyutilized to acquire predictive dosimetry for use in planning or ensuringsafety of subsequent RAIT studies.

Mullerian inhibiting substance (MIS) is a member of the transforminggrowth factor-β (TGFβ) superfamily of secreted protein hormones thatsignal through receptor complexes of type I and type II serine/threoninekinase receptors. The binding of MIS ligand to its receptor initiates asignaling cascade, including phosphorylation of Smad1, that is dependenton recruitment of type I receptors, ALK2 and ALK6, which also signal forbone morphogenetic proteins (Segev, D. L., et al. (2001) J. of Biol.Chem., 276:26799-26806). In males, MIS is produced in fetal andpostnatal testes. MIS binds to its receptor and triggers regression ofthe Mullerian ducts, the anlagen of the uterus, fallopian tubes andvagina (Hudson, P. L., at al. (1990) J. Clin. Endocrinol. Metab.,70:16-22). In contrast, MIS is not produced in females untiladolescence, thus allowing the above tissues to develop (Hudson, P. L.,et al. (1990) J. Clin. Endocrinol. Metab., 70:16-22).

In adult females, MIS type II receptor (MISIIR) is expressed on thesurface epithelium of the ovaries (Masiakos, P. T., et al. (1999) Clin.Cancer Res., 5:3488-3499). In mice, MISIIR mRNA has been detected inovarian surface epithelium (MOSE cells) and ovarian tissue (Connolly, D.C., et al. (2003) Cancer Res., 1389-1397). In rats, MISIIR mRNA has beendetected in embryonic, pubertal, and adult testes; the uterus; theovaries; and the embryonic lung (Teixeira et al. (1996) Endocinology,137:160-165; Catlin et al. (1997) Endocrinology, 138:790-796).Additionally, MISIIR has been detected in human cervical cancer cells,prostate cancer cells, breast epithelial cells, breast cancer celllines, breast fibroadenomas, breast tumors, and ductal carcinomas (Segevet al. (2000) J. Biol. Chem., 275:28371-28379; Segev et al. (2001) J.Biol. Chem., 276:26799-26806; Segev et al. (2002) Proc. Natl. Acad.Sci., 99:239-244; Barbie et al. (2003) Proc. Natl. Acad. Sci.,100:15601-15606).

Coelomic epithelium is the most common origin of human ovarian cancersand tumors of this origin express the MIS type II receptor. MISIIR mRNAis expressed in a number of ovarian carcinoma cell lines, includingOVCAR3, OVCAR5, OVCAR8, OV1063 and SKOV3 (Masiakos, P. T. et al., (1999)Clin. Cancer Res., 5:3488-3499). Furthermore, recombinant MIS boundtumor cells isolated from ascites in 15 of 27 (56%) ovarian cancerpatients and the binding of recombinant MIS to these tumor cells led tosignificant growth inhibition in 22/27 (82%) of these cases (Masiakos,P. T. et al., (1999) Clin. Cancer Res., 5:3488-3499). These findingsdemonstrate the relevance of MISIIR for anti-cancer therapies,particularly ovarian cancer.

SUMMARY OF THE INVENTION

In accordance with the present invention, novel antibody moleculeshaving specific binding affinity for the Mullerian Inhibiting SubstanceII Receptor (MISIIR) are provided. Such antibodies include, monoclonal,polyclonal, diabodies, tribodies, single domain antibodies, and scFv. Ina particular embodiment, the antibodies molecules are single chain Fvantibody molecules. In another embodiment of the invention, the singlechain Fv antibody molecules comprise an amino acid sequence selectedfrom the group consisting of SEQ ID NOS: 9-15.

In yet another embodiment, molecules comprising the single chain Fvantibody molecules of the invention are disclosed. Such moleculesinclude without limitation, a diabody, a tribody, a tetrabody, animmunotoxin, a recombinantly produced IgG, Fab, Fab′, F(ab′)₂, F(v),scFv, scFv₂, scFv-Fc, minibody, a bispecific antibody, an Affibody®, anda peptabody.

In a preferred embodiment, the antibody of the invention has bindingaffinity for the extracellular domain of MISIIR. More preferably, theantibodies bind MIS binding sites. Such sites include, for example, theamino acid sequences of SEQ ID NOS: 16-18.

According to another aspect of the invention, compositions and methodsfor treating cancer are provided wherein a patient is administered atherapeutically effective amount of the anti-MISIIR molecules of theinvention in a pharmaceutically acceptable carrier. In a particularembodiment of the invention, the cancer is selected from the groupconsisting of breast, prostate, cervical, ovarian, testicular, andpulmonary cancers. In a specific embodiment, the cancer is ovariancancer. In accordance with another aspect of the instant invention, theanti-MISIIR antibody molecule can be conjugated to at least of thefollowing agents, a chemotherapeutic agent, a radioisotope, a toxin, amagnetic bead, a detectable label and a pro-drug.

In yet another embodiment of the invention, the anti-MISIIR antibodiesare administered to a patient in combination with, prior to, or afteradministration of chemotherapeutic agents.

According to yet another aspect of the invention, compositions andmethods for imaging cancer, particularly ovarian cancer, are providedwherein a patient is administered a sufficient amount of an anti-MISIIRantibody molecule. In another embodiment, the anti-MISIIR antibody islabeled with a radioisotope and/or a contrast agent. The patient can bescanned by medical devices such as, without limitation, gamma cameras,mammography instruments, positron emission tomography (PET) cameras, andmagnetic resonance imaging (MRI) imaging.

In another embodiment of the instant invention, anti-MISIIR antibodiesare employed to detect the presence of MISIIR in patients suspected ofhaving cancer, particularly ovarian cancer. According to one aspect, theanti-MISIIR antibodies are employed as a diagnostic tool. In anotherembodiment of the instant invention, the presence of MISIIR is detectedin tissues and/or fluids, such as blood, from a patient to monitorprogression or remission of disease.

BRIEF DESCRIPTIONS OF THE DRAWING

FIG. 1 is an image of a Western blot of the purification of an MISIIRfusion protein. Proteins were separated by SDS-polyacrylamide gelelectrophoresis and detected by anti-His tag monoclonal antibodies.

FIG. 2 depicts the results of a PCR fingerprinting assay. Therestriction patterns of 10 clones were analyzed on an agarose gel.Unique patterns are noted in lanes 1-7, 9, and 10. M lane is molecularweight markers.

FIG. 3 is a graph of the eluted protein from the SuperDex75 sizeexclusion column on an HPLC system. Samples were taken over 30 minutesat intervals of 0.1 seconds. The large peak (of which most is above thefield of view) is labeled as the scFv of clone #7A.

FIG. 4 is an image of a polyacrylamide gel electrophoresis analysis ofthe proteins eluted from the size exclusion chromatography assay. Lanes1-5 are the elution fractions 12-16. Lane MW contains molecular weightstandards and the sizes of two of the molecular weight standards areindicated at the right of the gel.

FIG. 5 is a graph of the surface plasmon resonance assay performed withanti-MISIIR scFv (clone #17). MISIIR-Fc and IgG Fc were immobilized on aBIAcore® chip.

FIG. 6 is a schematic drawing of examples of the scFv-based antibodiesthat may be employed as anti-MISIIR antibodies.

FIG. 7 is a nucleotide sequence (SEQ ID NO: 8) encoding for the fusionof the extracellular domain of MISIIR and Fc.

FIGS. 8A and 8B are the nucleotide sequence (SEQ ID NO: 1) and aminoacid sequence (SEQ ID NO: 9) of the scFv antibody molecule of clone #17.

FIGS. 9A and 9B are the nucleotide sequence (SEQ ID NO: 2) and the aminoacid sequence (SEQ ID NO: 10) of the scFv antibody molecule of clone#23.

FIGS. 10A and 10B are the nucleotide sequence (SEQ ID NO: 3) and aminoacid sequence (SEQ ID NO: 11) of the scFv antibody molecule of clone#29.

FIGS. 11A and 11B are the nucleotide sequence (SEQ ID NO: 4) and aminoacid sequence (SEQ ID NO: 12) of the scFv antibody molecule of clone #7.

FIGS. 12A and 12B are graphs of the surface plasmon resonance of adiabody of clone #17 and an scFv-Fc of clone #7, respectively, on aBiacore® chip coated with MISIIR.

FIG. 13 is a graph of the binding of an scFv-Fc of clone #7 againstIGROV-1 cells. The filled in results are a control IgG.

FIGS. 14A and 14B are the nucleotide sequence (SEQ ID NO: 5) and theamino acid sequence (SEQ ID NO: 13) of the GY4 scFv antibody molecule.

FIGS. 15A and 15B are the nucleotide sequence (SEQ ID NO: 6) and theamino acid sequence (SEQ ID NO: 14) of the M2 scFv antibody molecule.

FIGS. 16A and 16B are the nucleotide sequence (SEQ ID NO: 7) and theamino acid sequence (SEQ ID NO: 15) of the M9 scFv antibody molecule.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, anti-MISIIR antibodies andmethods of use thereof are provided. Specifically, methods for theimmunodetection and imaging of cancer associated with MISIIR expressionand methods of treating the same are provided.

The antibodies of the invention include monoclonal, polyclonal, scFv andmolecules comprising a plurality of scFv. Also encompassed by thepresent invention are conjugates of the antibody molecules describedherein. Such conjugates include, without limitation, antibodies operablylinked to imaging reagents, contrast agents, chemotherapeutic agents,cytotoxic molecules (e.g., immunotoxins) and the like.

scFv molecules specific for human tumor-associated antigens have beenpreviously shown to be retained in a highly specific manner in humantumor xenografts growing in immunodeficient mice (Schier, R., et al.(1995) Immunotechnology, 1:63-71). Notably, quantitative tumor retentioncan be significantly enhanced by utilizing divalent scFv with a higheravidity for the antigen (Adams, G. P., et al. (1993) Cancer Res.,53:4026-4034). Additionally, the structure of the divalent scFv caninfluence the degree of localization in a tumor, with non-covalentdiabodies typically exhibiting four-fold greater localization at 24hours post injection that was seen with the gene-fused or chemicallyconjugated scFv dimers (Adams, G. P., et al. (1998) British J. Cancer,77:1405-1412).

In addition to avidity and structure, binding affinity can alsoinfluence the targeting properties of scFv molecules. Very high affinity(e.g., greater than 10⁻⁹ M) has been associated with reduced tumorlocalization and poor tumor penetration of scFv, likely due to asaturation of the antigen pool in the perivascular region of the tumor(Adams, G., et al. (1998) Cancer Res., 58:485-490; Adams, G. P., et al.(2001) Cancer Res., 61:4750-4755). In these studies, very high affinity(10⁻¹¹ M) anti-HER2/neu scFv molecules were observed byimmunofluorescence to only penetrate a few cell lengths from bloodvessels while low affinity (10⁻⁷ M or less) scFv targeting the sameantigenic epitope exhibited a diffuse penetration. Therefore, antibodymolecules with very high affinity may not be the most desirable forimmunotherapy with antibodies conjugated, e.g., to radioisotopes orchemotherapeutic agents as the conjugated antibodies may not penetrateefficiently enough in a short period of time. Unconjugated antibodymolecules (i.e. lacking radioactive particles or chemotherapeuticagents) with very high affinity may be preferred for therapy, however,as they have significantly longer time to penetrate into the tumor tokill target cells. FIG. 6 shows some of the constructs that can begenerated and used as anti-MISIIR antibodies. As noted herein, some ofthe divalent structures may exhibit improved retention or greaterinternalization, depending upon the affinity and the target epitope.

In a particular embodiment, the anti-MISIIR antibodies of the instantinvention are directed to specific regions of MISIIR (see, e.g., GenBankAccession Nos. NP_(—)065434 and NM_(—)020547). Specifically, theanti-MISIIR antibodies are directed to regions of MISIIR which interactwith MIS. These regions include, without limitation, amino acids 46-59(GTELPRAIRCLYSR; SEQ ID NO: 16), amino acids 92-109 (CDPSPRAHPSPGSTLFTC;SEQ ID NO: 17), and amino acids 81-101 (DSDEPGCESLHCDPSPRAHPS; SEQ IDNO: 18).

Exemplary amino acid sequences of anti-MISIIR antibodies of the instantinvention include SEQ ID NOS: 9-15. See FIGS. 8B-11B and FIGS. 14B-16B.Nucleic acid molecules encoding these antibodies are also encompassed bythe present invention. For example, SEQ ID NOS: 1-7.

I. DEFINITIONS

The following definitions are provided to facilitate an understanding ofthe present invention:

“Nucleic acid” or a “nucleic acid molecule” as used herein refers to anyDNA or RNA molecule, either single or double stranded and, if singlestranded, the molecule of its complementary sequence in either linear orcircular form. In discussing nucleic acid molecules, a sequence orstructure of a particular nucleic acid molecule may be described hereinaccording to the normal convention of providing the sequence in the 5′to 3′ direction. With reference to nucleic acids of the invention, theterm “isolated nucleic acid” is sometimes used. This term, when appliedto DNA, refers to a DNA molecule that is separated from sequences withwhich it is immediately contiguous in the naturally occurring genome ofthe organism in which it originated. For example, an “isolated nucleicacid” may comprise a DNA molecule inserted into a vector, such as aplasmid or virus vector, or integrated into the genomic DNA of aprokaryotic or eukaryotic cell or host organism.

When applied to RNA, the term “isolated nucleic acid” may refer to anRNA molecule encoded by an isolated DNA molecule as defined above.Alternatively, the term may refer to an RNA molecule that has beensufficiently separated from other nucleic acids with which it would beassociated in its natural state (i.e., in cells or tissues). An isolatednucleic acid (either DNA or RNA) may further represent a moleculeproduced directly by biological or synthetic means and separated fromother components present during its production.

With respect to single stranded nucleic acids, particularlyoligonucleotides, the term “specifically hybridizing” refers to theassociation between two single-stranded nucleotide molecules ofsufficiently complementary sequence to permit such hybridization underpre-determined conditions generally used in the art (sometimes termed“substantially complementary”). In particular, the term refers tohybridization of an oligonucleotide with a substantially complementarysequence contained within a single-stranded DNA molecule of theinvention, to the substantial exclusion of hybridization of theoligonucleotide with single-stranded nucleic acids of non-complementarysequence. Appropriate conditions enabling specific hybridization ofsingle stranded nucleic acid molecules of varying complementarity arewell known in the art.

For instance, one common formula for calculating the stringencyconditions required to achieve hybridization between nucleic acidmolecules of a specified sequence homology is set forth below (Sambrooket al., (1989) Molecular Cloning, Cold Spring Harbor Laboratory):

Tm=81.5° C.+16.6 Log [Na+]+0.41(% G+C)−0.63(% formamide)−600/#bp induplex

As an illustration of the above formula, using [Na+]=[0.368] and 50%formamide, with GC content of 42% and an average probe size of 200bases, the Tm is 57° C. The Tm of a DNA duplex decreases by 1-1.5° C.with every 1% decrease in homology. Thus, targets with greater thanabout 75% sequence identity would be observed using a hybridizationtemperature of 42° C. For example, hybridizations may be performed,according to the method of Sambrook et al., (supra) using ahybridization solution comprising: 5×SSC, 5×Denhardt's reagent, 1.0%SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.05% sodiumpyrophosphate and up to 50% formamide. Hybridization is carried out at37-42° C. for at least six hours. Following hybridization, filters arewashed as follows: (1) 5 minutes at room temperature in 2×SSC and 1%SDS; (2) 15 minutes at room temperature in 2×SSC and 0.1% SDS; (3) 30minutes-1 hour at 37.0 in 1×SSC and 1% SDS; (4) 2 hours at 42-65° in1×SSC and 1% SDS, changing the solution every 30 minutes.

The stringency of the hybridization and wash depend primarily on thesalt concentration and temperature of the solutions. In general, tomaximize the rate of annealing of the probe with its target, thehybridization is usually carried out at salt and temperature conditionsthat are 20-25° C. below the calculated Tm of the hybrid. Washconditions should be as stringent as possible for the degree of identityof the probe for the target. In general, wash conditions are selected tobe approximately 12-20° C. below the Tm of the hybrid. In regards to thenucleic acids of the current invention, a moderate stringencyhybridization is defined as hybridization in 6×SSC, 5×Denhardt'ssolution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42° C.,and washed in 2×SSC and 0.5% SDS at 55° C. for 15 minutes. A highstringency hybridization is defined as hybridization in 6×SSC,5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNAat 42° C., and washed in 1×SSC and 0.5% SDS at 65° C. for 15 minutes. Avery high stringency hybridization is defined as hybridization in 6×SSC,5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNAat 42° C., and washed in 0.1×SSC and 0.5% SDS at 65° C. for 15 minutes.

The term “probe” as used herein refers to an oligonucleotide,polynucleotide or DNA molecule, whether occurring naturally as in apurified restriction enzyme digest or produced synthetically, which iscapable of annealing with or specifically hybridizing to a nucleic acidwith sequences complementary to the probe. A probe may be eithersingle-stranded or double-stranded. The exact length of the probe willdepend upon many factors, including temperature, source of probe and useof the method. For example, for diagnostic applications, depending onthe complexity of the target sequence, the oligonucleotide probetypically contains 15-25 or more nucleotides, although it may containfewer nucleotides. The probes herein are selected to be complementary todifferent strands of a particular target nucleic acid sequence. Thismeans that the probes must be sufficiently complementary so as to beable to “specifically hybridize” or anneal with their respective targetstrands under a set of pre-determined conditions. Therefore, the probesequence need not reflect the exact complementary sequence of thetarget. For example, a non-complementary nucleotide fragment may beattached to the 5′ or 3′ end of the probe, with the remainder of theprobe sequence being complementary to the target strand. Alternatively,non-complementary bases or longer sequences can be interspersed into theprobe, provided that the probe sequence has sufficient complementaritywith the sequence of the target nucleic acid to anneal therewithspecifically.

The term “primer” as used herein refers to a DNA oligonucleotide, eithersingle-stranded or double-stranded, either derived from a biologicalsystem, generated by restriction enzyme digestion, or producedsynthetically which, when placed in the proper environment, is able tofunctionally act as an initiator of template-dependent nucleic acidsynthesis. When presented with an appropriate nucleic acid template,suitable nucleoside triphosphate precursors of nucleic acids, apolymerase enzyme, suitable cofactors and conditions such as a suitabletemperature and pH, the primer may be extended at its 3′ terminus by theaddition of nucleotides by the action of a polymerase or similaractivity to yield a primer extension product. The primer may vary inlength depending on the particular conditions and requirement of theapplication. For example, in diagnostic applications, theoligonucleotide primer is typically 15-25 or more nucleotides in length.The primer must be of sufficient complementarity to the desired templateto prime the synthesis of the desired extension product, that is, to beable anneal with the desired template strand in a manner sufficient toprovide the 3′ hydroxyl moiety of the primer in appropriatejuxtaposition for use in the initiation of synthesis by a polymerase orsimilar enzyme. It is not required that the primer sequence represent anexact complement of the desired template. For example, anon-complementary nucleotide sequence may be attached to the 5′ end ofan otherwise complementary primer. Alternatively, non-complementarybases may be interspersed within the oligonucleotide primer sequence,provided that the primer sequence has sufficient complementarity withthe sequence of the desired template strand to functionally provide atemplate-primer complex for the synthesis of the extension product.

Polymerase chain reaction (PCR) has been described in U.S. Pat. Nos.4,683,195, 4,800,195, and 4,965,188, the entire disclosures of which areincorporated by reference herein.

The terms “percent similarity”, “percent identity” and “percenthomology”, when referring to a particular sequence are used as set forthin the University of Wisconsin GCG software program.

The term “functional” as used herein implies that the nucleic or aminoacid sequence is functional for the recited assay or purpose.

“Natural allelic variants”, “mutants” and “derivatives” of particularsequences of nucleic acids refer to nucleic acid sequences that areclosely related to a particular sequence but which may possess, eithernaturally or by design, changes in sequence or structure. By closelyrelated, it is meant that at least about 75%, but often, more than 90%,of the nucleotides of the sequence match over the defined length of thenucleic acid sequence referred to using a specific SEQ ID NO. Changes ordifferences in nucleotide sequence between closely related nucleic acidsequences may represent nucleotide changes in the sequence that ariseduring the course of normal replication or duplication in nature of theparticular nucleic acid sequence. Other changes may be specificallydesigned and introduced into the sequence for specific purposes, such asto change an amino acid codon or sequence in a regulatory region of thenucleic acid. Such specific changes may be made in vitro using a varietyof mutagenesis techniques or produced in a host organism placed underparticular selection conditions that induce or select for the changes.Such sequence variants generated specifically may be referred to as“mutants” or “derivatives” of the original sequence.

The phrase “consisting essentially of” when referring to a particularnucleotide or amino acid means a sequence having the properties of agiven SEQ ID NO. For example, when used in reference to an amino acidsequence, the phrase includes the sequence per se and molecularmodifications that would not affect the basic and novel characteristicsof the sequence.

The term “promoters” or “promoter” as used herein can refer to a DNAsequence that is located adjacent to a DNA sequence that encodes arecombinant product. A promoter is preferably linked operatively to anadjacent DNA sequence. A promoter typically increases an amount ofrecombinant product expressed from a DNA sequence as compared to anamount of the expressed recombinant product when no promoter exists. Apromoter from one organism can be utilized to enhance recombinantproduct expression from a DNA sequence that originates from anotherorganism. For example, a vertebrate promoter may be used for theexpression of jellyfish GFP in vertebrates. In addition, one promoterelement can increase an amount of recombinant products expressed formultiple DNA sequences attached in tandem. Hence, one promoter elementcan enhance the expression of one or more recombinant products. Multiplepromoter elements are well-known to persons of ordinary skill in theart.

The term “enhancers” or “enhancer” as used herein can refer to a DNAsequence that is located adjacent to the DNA sequence that encodes arecombinant product. Enhancer elements are typically located upstream ofa promoter element or can be located downstream of or within a codingDNA sequence (e.g., a DNA sequence transcribed or translated into arecombinant product or products). Hence, an enhancer element can belocated 100 base pairs, 200 base pairs, or 300 or more base pairsupstream or downstream of a DNA sequence that encodes recombinantproduct. Enhancer elements can increase an amount of recombinant productexpressed from a DNA sequence above increased expression afforded by apromoter element. Multiple enhancer elements are readily available topersons of ordinary skill in the art.

The terms “transfected” and “transfection” as used herein refer tomethods of delivering exogenous DNA into a cell. These methods involve avariety of techniques, such as treating cells with high concentrationsof salt, an electric field, liposomes, polycationic micelles, ordetergent, to render a host cell outer membrane or wall permeable tonucleic acid molecules of interest. These specified methods are notlimiting and the invention relates to any transformation technique wellknown to a person of ordinary skill in the art.

A “replicon” is any genetic element, for example, a plasmid, cosmid,bacmid, phage or virus, that is capable of replication largely under itsown control. A replicon may be either RNA or DNA and may be single ordouble stranded.

A “vector” is a replicon, such as a plasmid, cosmid, bacmid, phage orvirus, to which another genetic sequence or element (either DNA or RNA)may be attached so as to bring about the replication of the attachedsequence or element.

An “expression operon” refers to a nucleic acid segment that may possesstranscriptional and translational control sequences, such as promoters,enhancers, translational start signals (e.g., ATG or AUG codons),polyadenylation signals, terminators, and the like, and which facilitatethe expression of a polypeptide coding sequence in a host cell ororganism.

The term “oligonucleotide,” as used herein refers to sequences, primersand probes of the present invention, and is defined as a nucleic acidmolecule comprised of two or more ribo- or deoxyribonucleotides,preferably more than three. The exact size of the oligonucleotide willdepend on various factors and on the particular application and use ofthe oligonucleotide.

The term “substantially pure” refers to a preparation comprising atleast 50-60% by weight of a given material (e.g., nucleic acid,oligonucleotide, protein, etc.). More preferably, the preparationcomprises at least 75% by weight, and most preferably 90-95% by weightof the given compound. Purity is measured by methods appropriate for thegiven compound (e.g. chromatographic methods, agarose or polyacrylamidegel electrophoresis, HPLC analysis, and the like).

The term “isolated protein” or “isolated and purified protein” issometimes used herein. This term refers primarily to a protein producedby expression of an isolated nucleic acid molecule of the invention.Alternatively, this term may refer to a protein that has beensufficiently separated from other proteins with which it would naturallybe associated, so as to exist in “substantially pure” form. “Isolated”is not meant to exclude artificial or synthetic mixtures with othercompounds or materials, or the presence of impurities that do notinterfere with the fundamental activity, and that may be present, forexample, due to incomplete purification, addition of stabilizers, orcompounding into, for example, immunogenic preparations orpharmaceutically acceptable preparations.

The term “gene” refers to a nucleic acid comprising an open readingframe encoding a polypeptide, including both exon and (optionally)intron sequences. The nucleic acid may also optionally includenon-coding sequences such as promoter or enhancer sequences. The term“intron” refers to a DNA sequence present in a given gene that is nottranslated into protein and is generally found between exons.

The phrase “operably linked,” as used herein, may refer to a nucleicacid sequence placed into a functional relationship with another nucleicacid sequence. Examples of nucleic acid sequences that may be operablylinked include, without limitation, promoters, cleavage sites,purification tags, transcription terminators, enhancers or activatorsand heterologous genes which when transcribed and translated willproduce a functional product such as a protein, ribozyme or RNAmolecule.

The phrase “solid support” refers to any solid surface including,without limitation, any chip (for example, silica-based, glass, or goldchip), glass slide, membrane, bead, solid particle (for example,agarose, sepharose, polystyrene or magnetic bead), column (or columnmaterial), test tube, or microtiter dish.

The phrases “affinity tag,” “purification tag,” and “epitope tag” mayall refer to tags that can be used to effect the purification of aprotein of interest. Purification/affinity/epitope tags are well knownin the art (see Sambrook et al., 2001, Molecular Cloning, Cold SpringHarbor Laboratory) and include, but are not limited to: polyhistidinetags (e.g. 6×His), polyarginine tags, glutathione-S-transferase (GST),maltose binding protein (MBP), S-tag, influenza virus HA tag,thioredoxin, staphylococcal protein A tag, the FLAG™ epitope, AviTagepitope (for subsequent biotinylation), dihydrofolate reductase (DHFR),an antibody epitope (e.g., a sequence of amino acids recognized andbound by an antibody), and the c-myc epitope.

A “cell line” is a clone of a primary cell or cell population that iscapable of stable growth in vitro for many generations.

An “immune response” signifies any reaction produced by an antigen, suchas a viral antigen, in a host having a functioning immune system. Immuneresponses may be either humoral in nature, that is, involve productionof immunoglobulins or antibodies, or cellular in nature, involvingvarious types of B and T lymphocytes, dendritic cells, macrophages,antigen presenting cells and the like, or both. Immune responses mayalso involve the production or elaboration of various effector moleculessuch as cytokines, lymphokines and the like. Immune responses may bemeasured both in in vitro and in various cellular or animal systems.Such immune responses may be important in protecting the host fromdisease and may be used prophylactically and therapeutically.

An “antibody” or “antibody molecule” is any immunoglobulin, includingantibodies and fragments thereof, that binds to a specific antigen. Theterm includes polyclonal, monoclonal, chimeric, single domain (Dab) andbispecific antibodies. As used herein, antibody or antibody moleculecontemplates recombinantly generated intact immunoglobulin molecules andimmunologically active portions of an immunoglobulin molecule such as,without limitation: Fab, Fab′, F(ab′)₂, F(v), scFv, scFv₂, scFv-Fc,minibody, diabody, tetrabody, single variable domain (e.g., variableheavy domain, variable light domain), bispecific, Affibody® molecules(Affibody, Bromma, Sweden), and peptabodies (Terskikh et al. (1997) PNAS94:1663-1668). Dabs can be composed of a single variable light or heavychain domain. In a certain embodiment of the invention, the variablelight domain and/or variable heavy domain specific for MISIIR areinserted into the backbone of the above mentioned antibody constructs.Methods for recombinantly producing antibodies are well-known in theart. For example, commercial vectors comprising constant genes to makeIgGs from scFvs are provided by Lonza Biologics (Slough, UnitedKingdom).

“Fv” is an antibody fragment which contains an antigen-recognition and-binding site. This region consists of a dimer of one heavy- and onelight-chain variable domain in tight, non-covalent association. It is inthis configuration that the three CDRs of each variable domain interactto define an antigen-binding site on the surface of the V_(H)-V_(L)dimer. Collectively, the six CDRs confer antigen-binding specificity tothe antibody. However, even a single variable domain (or half of an Fvcomprising only three CDRs specific for an antigen) has the ability torecognize and bind antigen, although often at a lower affinity than theentire binding site.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of an antibody, wherein these domains are present in asingle polypeptide chain. Generally, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the scFv to form the desired structure for antigen binding. Fora review of scFv see, for example, Pluckthun, A. in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) on thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Holliger et al.,(1993) Proc. Natl. Acad. Sci. USA, 90: 6444-6448.

With respect to antibodies, the term “immunologically specific” refersto antibodies that bind to one or more epitopes of a protein or compoundof interest, but which do not substantially recognize and bind othermolecules in a sample containing a mixed population of antigenicbiological molecules.

As used herein, the term “immunotoxin” refers to chimeric molecules inwhich antibody molecules or fragments thereof are coupled or fused(i.e., expressed as a single polypeptide or fusion protein) to toxins ortheir subunits. Toxins to be conjugated or fused can be derived formvarious sources, such as plants, bacteria, animals, and humans or besynthetic toxins (drugs), and include, without limitation, saprin,ricin, abrin, ethidium bromide, diptheria toxin, Pseudomonas exotoxin,PE40, PE38, saporin, gelonin, RNAse, protein nucleic acids (PNAs),ribosome inactivating protein (RIP), type-1 or type-2, pokeweedanti-viral protein (PAP), bryodin, momordin, and bouganin.

The term “conjugated” refers to the joining by covalent or noncovalentmeans of two compounds or agents of the invention.

Chemotherapeutic agents are compounds that exhibit anticancer activityand/or are detrimental to a cell (e.g., a toxin). Suitablechemotherapeutic agents include, but are not limited to: toxins (e.g.,saporin, ricin, abrin, ethidium bromide, diptheria toxin, Pseudomonasexotoxin, and others listed above; thereby generating an immunotoxinwhen conjugated or fused to an antibody); alkylating agents (e.g.,nitrogen mustards such as chlorambucil, cyclophosphamide, isofamide,mechlorethamine, melphalan, and uracil mustard; aziridines such asthiotepa; methanesulphonate esters such as busulfan; nitroso ureas suchas carmustine, lomustine, and streptozocin; platinum complexes such ascisplatin and carboplatin; bioreductive alkylators such as mitomycin,procarbazine, dacarbazine and altretamine); DNA strand-breakage agents(e.g., bleomycin); topoisomerase II inhibitors (e.g., amsacrine,dactinomycin, daunorubicin, idarubicin, mitoxantrone, doxorubicin,etoposide, and teniposide); DNA minor groove binding agents (e.g.,plicamydin); antimetabolites (e.g., folate antagonists such asmethotrexate and trimetrexate; pyrimidine antagonists such asfluorouracil, fluorodeoxyuridine, CB3717, azacitidine, cytarabine, andfloxuridine; purine antagonists such as mercaptopurine, 6-thioguanine,fludarabine, pentostatin; asparginase; and ribonucleotide reductaseinhibitors such as hydroxyurea); tubulin interactive agents (e.g.,vincristine, vinblastine, and paclitaxel (Taxol)); hormonal agents(e.g., estrogens; conjugated estrogens; ethinyl estradiol;diethylstilbesterol; chlortrianisen; idenestrol; progestins such ashydroxyprogesterone caproate, medroxyprogesterone, and megestrol; andandrogens such as testosterone, testosterone propionate,fluoxymesterone, and methyltestosterone); adrenal corticosteroids (e.g.,prednisone, dexamethasone, methylprednisolone, and prednisolone);leutinizing hormone releasing agents or gonadotropin-releasing hormoneantagonists (e.g., leuprolide acetate and goserelin acetate); andantihormonal antigens (e.g., tamoxifen, antiandrogen agents such asflutamide; and antiadrenal agents such as mitotane andaminoglutethimide). Preferably, the chemotheraputic agent is selectedfrom the group consisting of: placitaxel (Taxol®), cisplatin, docetaxol,carboplatin, vincristine, vinblastine, methotrexate, cyclophosphamide,CPT-11, 5-fluorouracil (5-FU), gemcitabine, estramustine, carmustine,adriamycin (doxorubicin), etoposide, arsenic trioxide, irinotecan, andepothilone derivatives.

The term “pro-drug” refers to a compound which is transformed in vivo toan active form of the drug. The pro-drug may be transformed to an activeform only upon reaching the target in vivo or upon internalization bythe target cell.

Radioisotopes of the instant invention include, without limitation,positron-emitting isotopes and alpha-, beta-, gamma-, Auger- and lowenergy electron-emitters. The radioisotopes include, without limitation:¹³N, ¹⁸F, ³²P, ⁶⁴Cu, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ⁶⁷Cu, ⁷⁷Br, ^(80m)Br, ⁸²Rb, ⁸⁶Y,⁹⁰Y, ⁹⁵Ru, ⁹⁷Ru, ^(99m)Tc, ¹⁰³Ru, ¹⁰⁵Ru, ¹¹¹In, ^(113m)In, ¹¹³Sn,^(121m)Te, ^(122m)Te, ^(125m)Te, ¹²³I, ¹²⁴I, ¹²⁵I, ¹²⁶I, ¹³¹I, ¹³³I,¹⁶⁵Tm, ¹⁶⁸Tm, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ^(195m)Hg, ²¹¹At, ²¹²Bi, ²¹³Bi, and²²⁵Ac. When the conjugated antibodies of the instant invention areemployed for radio-immunodetection, the radioisotope is preferably agamma-emitting isotope. When the conjugated antibodies of the instantinvention are employed for detection by ImmunoPET (positron emissiontomography), the radioisotope is preferably a positron-emitting isotopesuch as, without limitation, ¹³N, ¹⁸F, ⁸²Rb. When the conjugatedantibodies of the instant invention are employed for radioimmunotherapy(i.e., the treating of a patient with cancer), the radioisotope ispreferably selected from the group consisting of ⁹⁰Y, ¹³¹I, ¹⁷⁷Lu, and¹⁸⁶Re, although other radionuclides such as many of those listed aboveare also suitable.

The term “radiosensitizer”, as used herein, is defined as a moleculeadministered to animals in therapeutically effective amounts to increasethe sensitivity of the cells to radiation. Radiosensitizers are known toincrease the sensitivity of cancerous cells to the toxic effects ofradiation. Radiosensitizers include, without limitation,2-nitroimidazole compounds, and benzotriazine dioxide compounds,halogenated pyrimidines, metronidazole, misonidazole,desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, mitomycinC, RSU 1069, SR 4233, E09, RB 6145, nicotinamide, 5-bromodeoxyuridine(BUdR), 5-iododeoxyuridine (IUdR), bromodeoxycytidine,fluorodeoxyuridine (FudR), hydroxyurea, cisplatin, and therapeuticallyeffective analogs and derivatives of the same.

II. PREPARATION OF ANTIBODY MOLECULES

The antibody molecules of the invention may be prepared using a varietyof methods known in the art. Polyclonal and monoclonal antibodies areprepared as described in Current Protocols in Molecular Biology, Ausubelet al. eds. Antibodies may be prepared by chemical cross-linking, hybridhybridoma techniques and by expression of recombinant antibody fragmentsexpressed in host cells, such as bacteria or yeast cells. Indeed, Salhiet al. describe the generation of an anti-MISIIR monoclonal antibody,12G4, generated by a hybrid hybridoma technique (Biochem. J. (2004)379:785-793).

In one embodiment of the invention, the antibody molecules are producedby expression of recombinant antibody fragments in host cells. The genesfor several of the antibody molecules that target MISIIR have beencloned. The nucleic acid molecules encoding the MISIIR antibodyfragments are inserted into expression vectors and introduced into hostcells. The resulting antibody molecules are then isolated and purifiedfrom the expression system. The antibodies optionally comprise apurification tag by which the antibody can be purified.

The purity of the antibody molecules of the invention may be assessedusing standard methods known to those of skill in the art, including,but not limited to, ELISA, immunohistochemistry, ion-exchangechromatography, affinity chromatography, immobilized metal affinitychromatography (IMAC), size exclusion chromatography, polyacrylamide gelelectrophoresis (PAGE), western blotting, surface plasmon resonance andmass spectroscopy.

III. USES OF ANTI-MISIIR ANTIBODY MOLECULES

Anti-MISIIR antibodies have broad applications in therapy and diagnosis.Specifically, the anti-MISIIR antibody molecules of the invention may beused: (1) to directly alter the growth of tumors that express MISIIR;(2) to alter the growth of tumors that express MISIIR in combinationwith other cytotoxic agents; (3) to image tumors that express MISIIR;and (4) as a diagnostic tool.

1) The anti-MISIIR antibody molecules of the instant invention can beadministered to a patient in need thereof, as described hereinbelow. Theanti-MISIIR antibody molecules of the instant invention include theantibodies alone and antibodies conjugated to other agents such as,without limitation, chemotherapeutic agents, radioisotopes, pro-drugs,pro-drug activating enzymes capable of converting a pro-drug to itsactive form, and magnetic beads (see, for example, U.S. Pat. No.6,645,731). If the compound to be conjugated is proteinaceous, a fusionprotein may be generated with the antibody molecule. Radiosensitizersmay also be administered with the antibodies.

2) To alter the growth of tumors that express MISIIR, the anti-MISIIRantibody molecules of the instant invention may be administered to apatient in combination with other cytotoxic agents. These othercytotoxic agents include, without limitation, chemotherapeutic agents,external beam radiation, targeted radioisotopes, and other antibodies orsignal transduction inhibitors. Radiosensitizers may also beadministered with the antibodies.

3) When employed for imaging tumors, the anti-MISIIR antibody moleculesof the invention can be conjugated to radioisotopes as describedhereinabove. The anti-MISIIR antibody molecules can be conjugated to theradioisotopes by any method including direct conjugation and by linkingthrough a chelator (see, for example, U.S. Pat. No. 4,624,846). Theanti-MISIIR antibody molecules may also be conjugated to labels orcontrast agents such as, without limitation, paramagnetic orsuperparamagnetic ions for detection by MRI imaging and optical andfluorescence and/or mammography agents (examples of other labels areprovided in, for example, U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241). Paramagneticions include, without limitation, Gd(III), Eu(III), Dy(III), Pr(III),Pa(IV), Mn(II), Cr(III), Co(III), Fe(III), Cu(II), Ni(II), Ti(III), andV(IV). Fluorescent agents include, without limitation, fluorescein andrhodamine and their derivatives. Optical agents include, withoutlimitation, derivatives of phorphyrins, anthraquinones, anthrapyrazoles,perylenequinones, xanthenes, cyanines, acridines, phenoxazines andphenothiazines. Mammography agents include, without limitation,derivatives of iodine or metals such as gold, gold particles or goldnanoparticles.

In an alternative method, a secondary binding ligand, such as a secondantibody or a biotin/avidin ligand binding arrangement, which canrecognize the anti-MISIIR antibody molecules of the instant invention,may be conjugated with the agents described above instead of with theanti-MISIIR antibody molecules. The conjugated secondary binding ligandcan then be used in conjunction with anti-MISIIR antibody molecules inany of the assays described herein.

4) The anti-MISIIR antibody molecules of the invention may be used to 1)diagnose cancer in patient, 2) determine the prognosis of a patient,including stage and grade (particularly whether it is metastatic) of atumor and its potential sensitivity to therapy, 3) determine the originof a tumor, 4) determine the efficacy of a treatment of a patient. Inone embodiment the anti-MISIIR antibody molecules are utilized to detectthe presence of MISIIR in a biological sample from a patient. Thebiological sample may include biopsies of various tissues including,without limitation: breast, prostate, cervical, ovarian, testicular, andpulmonary. Cellular examples of biological samples include tumor cells,blood cells, ovarian cells, prostate cells, breast cells, testicularcells, cervical cells, and lung cells. The biological sample may also bea biological fluid, wherein shed MISIIR can be detected, such as,without limitation, blood, serum, nipple aspirate and urine. Manyimmunological assays are well known in the art for assaying ofbiological samples for the presence of a certain protein including,without limitation: immunoprecipitations, radioimmunoassays,enzyme-linked immunosorbent assays (ELISA), immunohistochemical assays,Western blot and the like.

The presence of MISIIR in fluids such as blood may be indicative of thepresence of cancer. The presence of MISIIR in fluids or sites not nearthe tumor may be indicative of metastases. The loss of MISIIR expressionin a patient, particularly one undergoing treatment, over time isindicative of remission (i.e., successful treatment) while the gain ofMISIIR expression in a patient over time can be indicative ofrecurrence. Additionally, the imaging techniques described hereinabovemay be employed to monitor the size of the tumor to determine theefficacy of a treatment. In a particular embodiment of the invention,other cancer diagnostic assays can be performed to confirm the resultsobtained with the instant invention.

The anti-MISIIR antibody molecules of the invention may also be used ingene therapy for direct targeting of vehicles (liposomes, viruses etc.)containing genes to specific tumors expressing MISIIR. In an exemplaryembodiment, liposomes may be studded by the anti-MISIIR antibodymolecules of the invention to facilitate tumor specific targeting. Inanother embodiment, anti-MISIIR antibodies may be expressed directly onthe surface of viruses or as fusions with viral coat proteins tofacilitate tumor specific targeting. The genes targeted in this mannercan have a direct anti-tumor effect, sensitize the tumor to other agentsor increase the susceptibility of the tumor to a host immune response.Anti-cancer agents such as chemotherapeutic agents, toxins, antibodies,antisense molecules, RNAi and/or radioisotopes may also be encapsulatedin liposomes so modified.

In another embodiment, the anti-MISIIR antibody molecules may be used todirect gene therapy vectors, including but not limited to modifiedviruses, to cells that express MISIIR. Viruses and other vectors mayalso be utilized to deliver the genes for the anti-MISIIR antibodymolecules to tumor cells where they could be produced and secreted intothe cellular microenvironment or, through the addition of additionalintracellular targeting sequences, they could be turned into intrabodiesthat localize to specific cellular compartments and knockout theexpression of their targets.

In yet another embodiment of the instant invention, the anti-MISIIRantibody molecules of the instant invention can be conjugated orcovalently attached to another targeting agent to increase thespecificity of the tumor targeting. Targeting agents can include,without limitation, antibodies, cytokines, and receptor ligands. In aparticular embodiment, the targeting agent is overexpressed on the tumoras compared to normal tissue. Additionally, the anti-MISIIR antibodymolecules of the instant invention can be conjugated or covalentlyattached to compounds which elicit an immune response such as, withoutlimitation, cytokines.

The present invention further encompasses kits for use in detecting theexpression of MISIIR in biological samples. Such kits may comprise theanti-MISIIR antibody molecules of the invention specific for MISIIR aswell as buffers and other compositions and instruction material to beused for the detection of the MISIIR.

IV. ADMINISTRATION OF ANTIBODIES

The antibodies as described herein will generally be administered to apatient as a pharmaceutical preparation. The term “patient” as usedherein refers to human or animal subjects. These antibodies may beemployed therapeutically, under the guidance of a physician for thetreatment of malignant tumors and metastatic disease.

The pharmaceutical preparation comprising the antibody molecules of theinvention may be conveniently formulated for administration with anacceptable medium such as water, buffered saline, ethanol, polyol (forexample, glycerol, propylene glycol, liquid polyethylene glycol and thelike), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents orsuitable mixtures thereof. The concentration of antibody molecules inthe chosen medium will depend on the hydrophobic or hydrophilic natureof the medium, as well as the size and other properties of the antibodymolecules. Solubility limits may be easily determined by one skilled inthe art.

As used herein, “biologically acceptable medium” includes any and allsolvents, dispersion media and the like which may be appropriate for thedesired route of administration of the pharmaceutical preparation, asexemplified in the preceding paragraph. The use of such media forpharmaceutically active substances is known in the art. Except insofaras any conventional media or agent is incompatible with the antibodymolecules to be administered, its use in the pharmaceutical preparationis contemplated.

The dose and dosage regimen of an antibody according to the inventionthat is suitable for administration to a particular patient may bedetermined by a physician considering the patient's age, sex, weight,general medical condition, and the specific condition and severitythereof for which the antibody is being administered. The physician mayalso consider the route of administration of the antibody, thepharmaceutical carrier with which the antibody may be combined, and theantibody's biological activity.

Selection of a suitable pharmaceutical preparation depends upon themethod of administration chosen. For example, the antibodies of theinvention may be administered by direct injection into any canceroustissue or into the surrounding area. In this instance, a pharmaceuticalpreparation comprises the antibody molecules dispersed in a medium thatis compatible with the cancerous tissue.

Antibodies may also be administered parenterally by intravenousinjection into the blood stream, or by subcutaneous, intramuscular orintraperitoneal injection. Pharmaceutical preparations for parenteralinjection are known in the art. If parenteral injection is selected as amethod for administering the antibodies, steps must be taken to ensurethat sufficient amounts of the molecules reach their target cells toexert a biological effect. The lipophilicity of the antibodies, or thepharmaceutical preparation in which they are delivered, may have to beincreased so that the molecules can arrive at their target locations.Furthermore, the antibodies may have to be delivered in a cell-targetingcarrier so that sufficient numbers of molecules will reach the targetcells. Methods for increasing the lipophilicity of a molecule are knownin the art. If a small form of the antibody is to be administered,including but not limited to a Fab fragment, a Dab, an scFv or adiabody, it may be conjugated to a second molecule such as, but notlimited to polyethylene glycol (PEG) or an albumin-binding antibody orpeptide to prolong its retention in blood.

Pharmaceutical compositions containing a compound of the presentinvention as the active ingredient in intimate admixture with apharmaceutical carrier can be prepared according to conventionalpharmaceutical compounding techniques. The carrier may take a widevariety of forms depending on the form of preparation desired foradministration, e.g., intravenous, oral or parenteral. In preparing theantibody in oral dosage form, any of the usual pharmaceutical media maybe employed, such as, for example, water, glycols, oils, alcohols,flavoring agents, preservatives, coloring agents and the like in thecase of oral liquid preparations (such as, for example, suspensions,elixirs and solutions); or carriers such as starches, sugars, diluents,granulating agents, lubricants, binders, disintegrating agents and thelike in the case of oral solid preparations (such as, for example,powders, capsules and tablets). Because of their ease in administration,tablets and capsules represent the most advantageous oral dosage unitform in which case solid pharmaceutical carriers are obviously employed.If desired, tablets may be sugar-coated or enteric-coated by standardtechniques. For parenterals, the carrier will usually comprise sterilewater, though other ingredients, for example, to aid solubility or forpreservative purposes, may be included. Injectable suspensions may alsobe prepared, in which case appropriate liquid carriers, suspendingagents and the like may be employed.

A pharmaceutical preparation of the invention may be formulated indosage unit form for ease of administration and uniformity of dosage.Dosage unit form, as used herein, refers to a physically discrete unitof the pharmaceutical preparation appropriate for the patient undergoingtreatment. Each dosage should contain a quantity of active ingredientcalculated to produce the desired effect in association with theselected pharmaceutical carrier. Procedures for determining theappropriate dosage unit are well known to those skilled in the art.

Dosage units may be proportionately increased or decreased based on theweight of the patient. Appropriate concentrations for alleviation of aparticular pathological condition may be determined by dosageconcentration curve calculations, as known in the art.

In accordance with the present invention, the appropriate dosage unitfor the administration of anti-MISIIR antibody molecules may bedetermined by evaluating the toxicity of the antibody molecules inanimal models. Various concentrations of antibody pharmaceuticalpreparations may be administered to mice with transplanted human tumors,and the minimal and maximal dosages may be determined based on theresults of significant reduction of tumor size and side effects as aresult of the treatment. Appropriate dosage unit may also be determinedby assessing the efficacy of the antibody molecule treatment incombination with other standard anti-cancer drugs. The dosage units ofanti-MISIIR antibody molecules may be determined individually or incombination with each anti-cancer treatment according to greatershrinkage and/or reduced growth rate of tumors.

The pharmaceutical preparation comprising the anti-MISIIR antibodymolecules may be administered at appropriate intervals, for example, atleast twice a day or more until the pathological symptoms are reduced oralleviated, after which the dosage may be reduced to a maintenancelevel. The appropriate interval in a particular case would normallydepend on the condition of the patient.

The following examples provide illustrative methods of practicing theinstant invention, and are not intended to limit the scope of theinvention in any way. While certain of the following examplesspecifically recite a certain type of anti-MISIIR antibody (e.g., scFvand diabody), the use of any anti-MISIIR antibody is within the scope ofthe instant invention. Additionally, while radioisotope conjugatedantibodies are also exemplified, immunotoxins may also be employed fortherapeutic purposes.

Example 1 Identification of MISIIR Specific Antibodies

Single-chain Fv (scFv) molecules specific for MISIIR were obtained byemploying a panning (selection) strategy of a human scFv combinatorialphage display library similar to that employed for identifying scFvmolecules specific for Her2/neu (Horak, E. M., et al. (2001) Proc. Amer.Assoc. Cancer Res., 39:774). Specifically, the MSIIR gene was isolatedby performing RT-PCR on a human testicle Poly (A) RNA library (Cat#7973; Ambion; Austin, Tex.). The portion of the resulting cDNA encodingthe ECD (extracellular domain) of MISIIR (FIG. 7) was cloned into thepSecTag2/Hygro vector (Invitrogen, Carlsbad, Calif.) which encodes for a6×His tag and a c-myc tag to be added to the carboxyl-terminus of theprotein and a secretion signal to be added to the amino-terminus. TheDNA encoding for the ECD of MISIIR was inserted in to the vector in twoorientations, either 5′ or 3′ of a human IgG1 Fc domain in order tofacilitate secretion of the fully folded MISIIR into the supernatant. Atobacco etch virus cleavage site was engineered into the constructbetween MISIIR and the Fc domain to facilitate the separation of thefinal product from the Fc domain.

HEK 293 cells, grown in serum-free media, were stably transfected withboth constructs and each fusion protein was isolated from culturesupernatants by immobilized metal affinity chromatography; IMAC(Hochuli, E., et al. (1988) Bio/Technology, 6:1321-1325). A western blotof the protein expressed from the construct encoding for the ECD ofMISIIR 5′ of the Fc domain is shown in FIG. 1. The purified fusionprotein was coated onto the sides of a Nunc-Immuno™ tube (Nalge Nunc;Rochester, N.Y.). A human scFv combinatorial phage display libraryconsisting of 10¹¹ unique scFv clones (provided by Wayne Marasco,Dana-Farber Cancer Institute) was mixed with 100-fold excess of humanIgG Fc domain and subsequently added to the Nunc-Immuno™ tube. Thepremixing of the library with a large excess of Fc domain diminished thelikelihood that phage expressing anti-Fc scFv would bind the immobilizedMISIIR-Fc fusion protein. Selection of phage displaying anti-MISIIR scFvmolecules was performed similarly to the method described in Adams andSchier (Adams, G. P. and Schier, R. (2000) Methods in Molecular MedicineVol. 39: Ovarian Cancer. Ed. Bartlett, J. The Humana Press, Totowa,N.J., pp. 729-747). Briefly, the Nunc-Immuno™ tube containing the phagelibrary and excess Fc domain was washed and reactive phage were elutedby the addition of 100 mM triethylamine. TG1 E. coli were challengedwith the eluted phage. Subsequently, phage particles were isolated fromthe infected bacteria and employed in subsequent rounds of binding andselecting.

After three rounds of selection, 200 phage-scFv clones were randomlyisolated from the selection plates containing thousands of colonies. Theclones were expanded and assayed by ELISA for binding to MISIIR-Fc andto IgG Fc domain. Thirty-three clones exhibited significant specificityfor MISIIR-Fc without any evidence for specificity for IgG Fc domain.

After isolation of the vector containing the scFv gene from the 33 phageclones by phenol chloroform extraction, a PCR fingerprinting assay wasperformed. Briefly, the PCR fingerprinting assay entailed digesting theisolated scFv gene with BstN1 at 60° C. for 2 hours and analyzing therestriction pattern on an agarose gel. As exemplified in FIG. 2, 16 ofthe clones had different restriction patterns and therefore containedscFv genes of unique nucleotide sequences.

DNA sequencing of the clones determined to be unique by PCRfingerprinting revealed that 12 of the 16 clones possessed unique aminoacid sequences. All 12 unique clones were transferred into the pCYNexpression vector (also referred to as pUC119mychis), expressed in E.coli with induction by the addition ofisopropyl-β-D-thioglactopyranoside (IPTG), and then purified by IMAC(Schier, R., et al. (1995) Immunotechnology, 1:73-81; Adams, G. P., etal. (2000) Methods in Molecular Medicine Vol. 39:Ovarian Cancer, TheHumana Press: Totowa, N.J., 729-747). The protein eluted from the resinwas further purified by size exclusion chromatography using HPLC (highperformance liquid chromatography) on a Superdex™ 75 column (Amersham;Piscataway, N.J.) (FIG. 3). The elution pattern indicated thatanti-MISIIR scFv from clone #7A is primarily monomeric. 2.5 mg ofpurified anti-MISIIR scFv from clone #7A (major peak in FIG. 3) wasobtained from 1 liter of bacterial culture. Polyacrylamide gelelectrophoresis analysis of the elution fractions from the sizeexclusion chromatography demonstrated the sequential purification schemeresulted in highly purified product (FIG. 4).

All 12 purified scFv molecules were also evaluated by surface plasmonresonance on a BIAcore® (Uppsala, Sweden) instrument to determine theability of the scFv molecules to bind highly purified MISIIR ECD.Surface plasmon resonance analysis revealed that at least 7 of the 12scFv molecules bind to MISIIR-Fc but not to IgG alone (see, e.g., clone#17 (FIG. 8), which displayed a high affinity for the ECD of MISIIR, inFIG. 5). As seen in FIG. 5, the dissociation rate of scFv from MISIIR-Fcis slow, therefore indicating a strong interaction between the antibodyand MISIIR. Such a strong interaction will allow for more durableretention of the antibody to a tumor expressing MISIIR.

The nucleotide sequences of certain scFv clones which displayed specificbinding to MISIIR are provided in FIGS. 8-11, 14, 15 and 16 (SEQ ID NOs1-7).

Surface plasmon resonance (SPR) technology was used to determine thebinding of the anti-MISIIR molecules to MISIIR ECD. The SPR responsereflects a change in the mass concentration as molecules bind ordissociate from an antigen immobilized on a sensor chip. These studieswere performed on a BIAcore 1000 instrument. The MISIIR-Fc ECD fusionprotein target antigen was coated on a CM5 chip at a concentration of5,000 resonance units (RU). The anti-MISIIR antibody molecules were thenflowed over the chip and the difference in the response units (RU)between the start point and the end was determined. A negative controlprotein (Fc) was coated on a chip at 4,800 RU and was used to rule outbinding to the Fc portion of the MISIIR-Fc fusion protein. Theassociation and dissociation rates, and the affinity of each moleculefor MISIIR ECD were determined by passing serially diluted samples overan MISIIR-Fc ECD coated chip with a lower concentration of targetantigen (600 RU) as per the BIAcore manufacturer's instructions. Theevaluation of the data was done using BIAEvaluation 3.0 software. FIG.12A shows the SPR of a diabody of clone #17 and FIG. 12B shows the SPRof an scFv-Fc of clone #7. Additionally, an scFv of clone #7 and an scFvof clone #17 demonstrated an affinity for MISIIR ECD by SPR of 3×10⁻⁷ Mand 0.9×10⁻⁷ M, respectively.

The binding of the antibodies was also tested in a standardfluorescence-activated cell sorter (FACS) assay. IGROV-1 cells, whichexpress MISIIR, were analyzed for binding to an scFv-Fc of clone #7 anda control IgG. Binding of the scFv-Fc to the cells was detected with afluorescin conjugated goat anti-human IgG (Catalog #AHI0408; BiosourceInternational, Camarillo, Calif.). FIG. 13 demonstrates specific bindingof the scFv-Fc of clone #7 for the MISIIR expressing IGROV-1 cells.

Example 2 Identification of MISIIR Antibodies with Cytotoxic orAnti-Proliferative Effects

MIS has been reported to exhibit antiproliferative effects in manyovarian cancer cell lines (Masiakos, P. T., et al., (1999) Clin. CancerRes., 5:3488-3499). Accordingly, some anti-MISIIR scFv molecules can beexpected to exhibit similar antagonistic effects. Therefore, theisolated unique anti-MISIIR scFv molecules identified in Example 1 canbe evaluated for antiproliferative and pro-apoptotic effects on ovariantumor cells that overexpress MISIIR and on control cells that lackMISIIR. Notably, scFv molecules bind to antigen monovalently, butengineered antibody-based molecules may also bind divalently. Therefore,scFv molecules can also be assayed as divalent antibodies in each of theassays described herein by contacting the anti-MISIIR scFv moleculeswith an anti-myc antibody such as 9E10 antibody (Santa CruzBiotechnology; Santa Cruz, Calif.). Inasmuch as the anti-MISIIR scFvmolecules contain a myc tag expressed on the carboxyl terminus, thepresence of the anti-myc antibodies generates dimeric MISIIR binders.Additionally, recombinant MIS or purified MIS can be included in eachassay as a positive control.

An example of an assay that may be employed to determine the effects ofthe anti-MISIIR scFv molecules on the growth and viability of cells isan MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide)assay (see, e.g., Sigma, St. Louis, Mo.). Briefly, living cells are ableto metabolize MTT and the reaction product can be monitoredcolormetrically. The MTT assay may be performed as previously describedwherein the purified anti-MISIIR scFv molecules are employed over a widerange of dilutions (e.g., ranging from 1 μM to 20 pM) in order toidentify optimal doses (Amoroso, A. R., et al. (1996) Cancer Research,56:113-120).

scFv clones that inhibit the proliferation of cell lines that expressMISIIR (e.g., OVCAR-3, SK-OV-3, IGROV-1, and MOVAR) without altering thegrowth of cell lines that lack MISIIR (e.g., COS cells) then will beassayed in clonogenicity assays designed to evaluate their ability toinduce cytotoxicity of the cell lines (Connolly, D. C., et al. (2003)Cancer Res., 1389-1397). Briefly, SK-OV-3, OVCAR-3, IGROV-1, MOVCAR andCOS cells can be plated at a low concentration in tissue culture platesin fresh media or conditioned media in the presence of increasingconcentrations of anti-MISIIR scFv. The cells can then be grown for oneweek at 37° C. Colonies can then be stained with, for example, 0.25%Methylene Blue in 30% ethanol and the number of colonies present in thetreated plates can be compared to those in untreated plates. Once aresponse curve is determined, assays can be repeated with the additionof increasing doses of gamma irradiation using, for example, a cesiumirradiator system in order to look for additive or synergistic effectsof the anti-MISIIR scFvs and radiation. As noted above, the assays canbe performed using both monovalent anti-MISIIR scFv and anti-MISIIR scFvthat have been crosslinked with the anti-myc tag 9E10 MAb.

The anti-MISIIR scFv molecules that mediate cytotoxic effects in theclonogenicity assays can also be evaluated for the ability to induceapoptosis in cells that overexpress MISIIR. As an example, an ELISAassay can be performed to detect the translocation of phosphatidylserinefrom the inner face of the plasma membrane to the cell surface, which isassociated with early apoptotic events. These assays can be performedusing, for example, the ApoAlert™ Annexin V Apoptosis Kit (Clontech,Palo Alto, Calif.), which employs an ELISA assay based upon the highaffinity of annexin V for phosphatidylserine. Briefly, at various timesafter incubation with anti-MISIIR scFv, MISIIR positive and negativecells can be washed, incubated with the annexin V-FITC conjugate, andevaluated in a microtiter plate reader. As above, the specificity of theresults can be based upon the observation of a positive signal in thewells containing, for example, SK-OV-3, OVCAR-3, and possibly MOVCARcells (depending upon epitope conservation) and no change in the signalin the wells containing negative controls such as COS cells which lackMISIIR. Alternatively, TUNEL (terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end-labeling) assays can be performed ontarget and control cells after 24 hrs in the presence or absence ofanti-MISIIR scFv (Surh, C. D. and Sprent, J. N. (1994) Nature,372:100-103).

Example 3 Diabody and scFv-Fc Construction

Diabodies can be created from the anti-MISIIR scFv genes using, forexample, the same techniques as employed to create the anti-HER2/neudiabody from the C6.5 scFv (Adams, G. P. et al. (1998) British J.Cancer, 77:1405-1412). Diabodies can be generated from the scFv clonesthat had the greatest affect on the growth/survival properties of tumorcells and from clones that did not alter these characteristics ascontrols. As described, each diabody will be composed of two identicalhomodimers, but bispecific heterdiabodies can also be prepared. Briefly,the V_(H) and V_(L) genes from each selected scFv can be amplified fromthe scFv DNA and gel purified.

The V_(H) and V_(L) genes can be joined together by, for example, PCRsplicing by overlap extension using an oligonucleotide which encodes a 5amino acid linker (G₄S) between the C-terminus of the V_(H) and theN-terminus of the V_(L). Indeed, the diabody of clone #17 was generatedwith this G4S linker. The primer employed to amplify the variable heavygene from the 3′ end encoded for three extra glycines and contained aBamHI site. The primer employed to amplify the variable light gene fromthe 5′ end contained a BamHI site. Notably, the BamHI site introduced aglycine-serine sequence into the encoded sequence for the antibody whenthe PCR fragments were ligated together, thereby resulting in the G4Slinker.

The diabody gene can be cloned into a vector such as pUC119Sfi-NotmycHis (Adams, G. P., et al. (1998) British J. Cancer,77:1405-1412), resulting in the addition of the myc peptide tag (Schier,R., et al. (1995) Immunotechnology, 1:73-81) followed by ahexa-histidine tag at the C-terminal end of the diabody. The vector alsoencodes the pectate lyase leader sequence, which directs expression ofthe diabody into the bacterial periplasm where the leader sequence iscleaved. This makes it possible to harvest native properly foldeddiabody directly from the bacterial periplasm. Native diabody can beexpressed and purified from the bacterial periplasm using, for example,immobilized metal affinity chromatography (IMAC) followed by FPLC sizeexclusion chromatography using a Superdex 75 column (Breitling, S. D.,et al. (1991) Gene 104:147-153; Hochuli, E., et al. (1998)Bio/Technology, 6:1321-1325). Each diabody can be assayed as describedabove for the scFv molecules to determine the ability of each diabody toalter the growth properties of cells that overexpress MISIIR.

The scFv-Fc of clone #7 was generated using the hinge stuffer vectorsystem (see, for example, www.nfcr-ctae.org). Briefly, the hinge stuffervector is a pcDNA3.1 based plasmid in which a chimeric human F105 VHleader sequence (with 3′ pelB leader) and human IgG1 sequence (hinge,CH2 and CH3 domains) flank SfiI (5′) and Not1 (3′) cloning sites thatcontain stuffer DNA (circa 439 bp). Upon digestion of the plasmid withSfiI and NotI restriction enzymes, the stuffer DNA drops out and thescFv fragments with identical restriction sites are cloned in frame. Inthis way, the bivalent human sFv-IgG fusion proteins are expressed andsecreted from mammalian cells.

Example 4 In Vivo Distribution Assays

Diabodies generated in Example 3 can be radiolabeled with ¹²⁵I andtested for their ability to target human ovarian carcinoma xenograftsgrowing in immunodeficient mice. Notably, ¹¹¹In, among other isotopes,can be used in place of ¹²⁵I, particularly if radioiodine labeleddiabodies are rapidly internalized. In particular, in vivobiodistribution and pharmacokinetics can be determined and cumulativetumor:organ ratios can be calculated. These values can be used in MIRD(Medical Internal Radiation Dose) dosimetry calculations to determinethe potential dose that would be achieved with the therapeutic isotopes13 and ⁹⁰Y in place of ¹²⁵I. Such assays can be performed in SCID miceas previously described (Adams, G. P., et al. (1993) Cancer Res.,53:4026-4034). Briefly, groups of mice can receive intravenous (i.v.)injections of radioiodinated diabody and blood samples can be collectedserially by retro-orbital bleeding at, for example, 5, 15, 30, 45minutes, 1, 2, 4, 8, 16, 24, 48 and 72 hours post administration. Thequantity of radioisotope retained per ml of blood can be determined.Trichloracetic acid (TCA) precipitation assays (Adams, G. P., et al.(1995) J. Nucl. Med., 36:2276-2281) can be performed to differentiatebetween protein-associated and unassociated counts to ensure that thedistribution of the diabody is being followed. The pharmacokinetics canthen be determined by analysis using the Rstrip program (Micromath, SaltLake City, Utah). Biodistribution studies can also be performedsimilarly except that the SCID mice will also bear establishedsubcutaneous 100-200 mm³ tumors, such as OVCAR-3 or SK-OV-3 tumors, thatoverexpress MISIIR (Masiakos, P. T., et al. (1999) 5:3488-3499). AUC(area under the curve) calculations can then be determined from % ID(injected dose)/g values using, for example, the NCOMP program (Laub, P.and J. Gallo (1996) J. Pharm. Sci., 85:393-395).

Dose estimates for radiolabeled antibodies in humans can be calculatedusing mouse biodistribution data from the same molecule based upon thefollowing assumptions: 1) tumor and normal organ concentration data inthe mouse can be extrapolated to “reference man”; 2) thepharmacokinetics and biodistribution of ¹¹¹Indium-radiolabeled antibodyare equal to those of antibodies radiolabeled with ⁹⁰Yttrium; and 3) theresidence time of the radiopharmaceutical in tumors and organs in man isthe same as that in the animal model. Finally, the S values (indicatorof the radioactive doses that are both absorbed and emitted by a givenorgan; see MIRD program) for the mouse organs must be normalized to thehuman organs based upon the size of the organ relative to the wholeanimal versus the size of the organ relative to the whole person.

All dose estimates can be calculated based on Formula A([RT/PIDRB]_(man)=[RT/PIDRB]_(mouse)) where RT=AUC/Activity administeredand PIDRB=Percent Injected Dose Remaining in the Body. The computerprogram MIRD DOSE 3 (supplied by Oak Ridge Associated Universities) canbe used to calculate radiation absorbed doses to all the organs in“reference man” based on the residence times established in the aboveequation. Since there are no S values available for tumors, the dose totumor can be calculated based on separating the dose contributed by βparticles and photons (Formula B: dose from β particles=D_(np)=([0.408 gcGy/μCi h]/[Tumor mass (g)])×Accumulated Activity (μCi h)). The dosefrom photons (D_(p)) can be counted equivalent to fraction of total bodydose calculated for photons only (D_(tumor)=D_(np)+D_(p)). Molecules canbe selected for evaluation in preclinical therapy trials based uponpredicted human dosimetry from the MIRD calculations and tumor:normalorgan AUC ratios. Preferably, diabodies will exhibit highly specifictumor targeting, e.g., a 3:1 ratio of cumulative tumor:normal dosimetry.Particular emphasis can be placed upon the LD₅₀ values formyelosuppression-sensitive organs such as the bone marrow (200-300 cGy)and those believed to be involved in the catabolism of the radiolabeledprotein such as the kidneys (1500 cGy) and liver (4000 cGy). Diabodiesselected for therapy studies can have predicted delivery of more than4000 cGy to tumor, with an associated normal organ dose limit of lessthan 200 cGy for the bone marrow, 1200 cGy for kidneys and 3000 cGy tothe liver (Murray, J., et al. (1994) Cancer, 73:1057-1066).

Example 5 Imaging Studies

Ovarian cancers poses unique challenges for early detection and inmonitoring responses to therapy (Ozols, R. F., et al. (2001) Cancer:Principles and Practice of Oncology, Lippincott Williams and Wilkins:Philadelphia, Pa.). CA-125 antigen detection in the circulating bloodhas proven useful in assessing the overall benefits of surgery orchemotherapy (Skates, S. J., et al. (2000) Methods in Molecular MedicineVol. 39: Ovarian Cancer, The Humana Press: Totowa, N.J.). Non-invasiveimaging by CT scanning, however, is insufficiently sensitive or specificto assess the impact of surgery or identify sites of tumor progressionor regression. Antibody-based imaging of ovarian cancer has employed avariety of antibodies and radionuclide pairs (Kalofonos, H. P., et al.(1999) Acta Oncologica, 38:629-634), but none of these approaches hasemerged as being sufficiently sensitive and specific for routine use.

The utility of using an anti-MISIIR diabody as a vehicle for thedetection of ovarian cancer can be compared with the images obtainedusing the current PET imaging agent ¹⁸FDG (¹⁸F fluorodeoxyglucose). Anexample of an assay for testing the sensitivity of the anti-MISIIRdiabodies for this purpose is as follows.

Due to the significantly longer physical half-life of ¹²⁴I, ¹⁸FDGimaging can be performed before conjugated diabody imaging studies.Immunodeficient mice bearing established 300 mm³ subcutaneous (s.c.)human SK-OV-3 or OVCAR-3 ovarian carcinoma tumors can be administered 50μCi of ¹⁸FDG by i.v. tail vein injection. At one and two hours afterinjection, the mice can be anesthetized and imaged on a Discovery™ LSPET/CT camera (GE Medical Systems; Waukesha, Wis.). Twenty four hourslater, the diabody that is determined to be associated with the greatestdegree of tumor targeting specificity in the biodistribution/dosimetrystudies described above can be labeled with ¹²⁴I, a positron-emittingradioisotope with a 4 day half-life) using the ⁶⁴Cu, Chloramine Tmethod. Notably, among other isotopes, can be used in place of ¹²⁴I forimaging studies, particularly if the radioiodine-labeled diabodies arerapidly internalized. The ¹²⁴I-conjugated diabody (50 microCuries on 50micrograms of diabody per mouse) can be administered by i.v. tail veininjection and the mice can be anesthetized as described above. The micecan be imaged at, for example, 4, 12, 24, 36 and 48 hours postadministration. Imaging can be performed, for example, on the Discovery™LS Whole Body PET/CT Scanner (GE Medical Systems) or microPET®(ConcordeInstruments; Knoxyille, Tenn.).

The standard clinical PET/CT acquisition protocol may be significantlymodified for murine imaging. A custom-designed platform can be mountedon to the edge of imaging table for imaging up to 3 mice simultaneously.The current setting for CT (computed tomography) can be reduced to 50mAs and PET data can be acquired in 2D mode for 10 minutes. PET imagescan be reconstructed using a standard manufacturer supplied OSEMalgorithm with 28 subsets and 2 iterations, with a reduced display fieldof view of 30 cm diameter, in a 256×256 matrix. CT can be used forapplying attenuation correction. PET activity data can be corrected forradioactive decay and normalized for the dose administered. AdditionalCT images can also be acquired with a slice thickness of 1 mm. Imagescan be reviewed on an eNTEGRA work station (GE Medical Systems) as 3-Dvolume sets with simultaneous display of transverse, coronal andsagittal panes. Regions of interest (ROI's) can be drawn by twoindependent observers around tumors using a combination of PET and CTinformation to identify tumor edges. Both the maximum and average tumoractivity concentration can be determined for any observed tumors fromPET images. The size of the tumor can be estimated from CT images andactivity can be corrected for object size effect using an experimentallydetermined recovery coefficient measured from mouse phantom studies with¹²⁴I.

The specificity of anti-MISIIR diabodies, degree of tumor targeting andretention as compared to normal tissue, and estimation of tumor andnormal tissue dosimetry associated with radiolabeled anti-MISIIRdiabodies can also be determined in humans. An example of such a studyfollows.

Women with metastatic, measurable MISIIR expressing ovarian cancer canbe eligible for this study. MISIIR expression can be determined by IHC(immunohistochemistry) on archived biopsy or aspirate specimens (Garciade Palazzo, I., et al. (1993) Intl. J. Biol. Markers, 8:233-239; Weiner,L. M., et al. (1995) Cancer Res., 55:4586-4593). Eligible patientsshould have acceptable normal organ function and should provide writteninformed consent. Prior antibody therapy can be permissible providedthat at least two months have elapsed from the last antibody infusion.

On Day One, patients can receive an intravenous injection of 10 mCi of¹⁸FDG. One hour later, PET/CT fusion images will be acquired using aDiscovery™ LS PET/CT instrument (GE Medical Systems) or equivalentimaging system. On Day Two or Three, the patients can receive 5 mCi (180MBq) of ¹²⁴I on an escalating dose of anti-MISIIR diabody (5, 10, 20, 30or 40 mg) by i.v. bolus injection. Whole body PET/CT images can beacquired and blood samples also can be obtained at about one hour, 4-8hours, 24 hours, 48 hours, 72 hours and 120 hours after administration.Regions of interest can be drawn around critical organs and the amountof localized activity can be determined. Blood samples can be countedalong with standards in a gamma well counter to determine the activityremaining in circulation. The projected radiation exposure to each organand to bone marrow that would have been associated with a therapeuticdose if ¹³¹I was used in place of ¹²⁴I can then be determined using theMIRD Dose 3 software package (Oak Ridge Associated Universities).

Safety can be determined by the ongoing monitoring of the incidence andintensity of adverse events graded by the Common Toxicity Criteria.Following injection of the radiopharmaceuticals, whole body PET/CTimaging can be performed immediately at the time points indicated above.As the diabody is a novel construct, regular blood samples can beobtained to assay for the development of human anti-diabody antibodies(HADA). These data can be used to obtain dosimetry estimates todetermine if ¹³¹I labeled anti-MISIIR diabody would be associated with apredicted therapeutic effect and could be safely administered topatients. If so, this can provide justification for proceeding to aseparate radioimmunotherapy clinical trial employing the ¹³¹I labeledanti-MISIIR diabody.

Statistical analysis of the results of this study can be performed asfollows. Briefly, logistic regression can be conducted to model theprobability of DLT (dose-limiting toxicity) or of efficacious responseas a function of protein dose. Polynomial functions of dose can beconsidered to permit the possibility of non-monotone dependence on dose(e.g., maximal efficacy achieved at a dose strictly less than themaximum administered in the study). The logistic regression models canprovide an estimate of the lowest protein dose such that (1) theestimated percentage of patients expected to exhibit favorable dosimetryis at least 50% and (2) the estimated prevalence of DLT is no greaterthan 10%. Generalized estimating equations in the context of logisticregression can be used to model the probability of a positive diagnosis(i.e., sensitivity) as a function of protein dose and image agent (i.e.,compare ¹⁸FDG and multiple ¹²⁴I-based images).

Example 6 Therapy Studies

The ability of unconjugated diabody to treat ovarian cancer xenograftscan be determined. If available, diabodies that either inhibit or do notsuppress in vitro tumor cell growth can be employed as controls in thesestudies. An example of such studies is described briefly here. 8-12 weekold SCID mice can receive s.c. implants of 5×10⁶ OVCAR-3, IGROV-1, orSK-OV-3 ovarian cancer cells. After the tumors have grown toapproximately 100 mm³, cohorts of about 10 mice can be treated dailywith four escalating dose levels, which can be determined based upon theresults of the in vitro studies described hereinabove, of the diabodyclone that exhibited the greatest efficacy in the in vitro growthinhibition studies described hereinabove. The mice can be observed fortumor growth and treatment-related toxicity by noting, e.g., weight lossand distress (Adams, G. P., et al. (2000) Nuc. Med. and Biol.,27:339-346). Tumors can be measured (length×width×depth) with calipersabout three times per week and the total volume can be calculated usingthe ellipsoidal method (Hann, H. W. L., et al. (1992) Cancer,70:2051-2056). If these results indicate that the unconjugated diabodyis associated with an in vivo anti-tumor effect, the V_(H) and V_(L)domains can be incorporated into a human IgG1 molecule to potentiallyafford greater bioavailability and the therapeutic potential of theresulting anti-MISIIR IgG can be assessed.

The ability of the anti-MISIIR diabody and IgG to treat early,spontaneously occurring ovarian tumors in the MISIIR-TAg transgenicmouse model can also be assessed. Briefly, the MISIIR-TAg transgenicmouse line expresses the transforming region (TAg) of simian virus 40(SV40) under the control of the MISIIR promoter (Connolly, D. C., et al.(2003) Cancer Res., 1389-1397). Approximately 50% of the femaleMISIIR-TAg mice develop ovarian tumors between 6 and 13 weeks of age.These ovarian tumors, in addition to a cell line, MOVCAR, derived fromthe ascites of the MISIIR-TAg mice, express endogenous MISIIR and TAg asdetermined by RT-PCR (Connolly, D. C., et al. (2003) Cancer Res.,1389-1397). The ability of the anti-MISIIR diabody and IgG to treat theovarian tumors in MISIIR-TAg mice can be determined by the followingexample. Briefly, cohorts of approximately 10 MISIIR-TAg mice can betreated with unconjugated anti-MISIIR diabody or IgG or left untreatedat about 6, 9 and 12 weeks of age. The mice can be euthanized anddissected when approximately 30-50% of the untreated control micedisplay evidence of tumor formation (e.g., ascities). After documentingevidence of gross lesions, the tissues can be fixed in formalin andprocessed for histopathological examination. The significance of therapyefficacy with unconjugated anti-MISIIR diabody and IgG can be determinedby comparing tumor growth in the presence of the agent compared to tumorgrowth in the controls.

The anti-MISIIR antibodies can also be tested in radioimmunotherapystudies as exemplified herein. Diabody molecules typically displayseveral characteristics such as rapid renal elimination, more efficienttumor penetration and higher binding avidity that frequently makediabodies superior to other antibody formats, such as intact IgG,(scFv)₂ and scFv, in radioimmunotherapy. Therefore, the diabodyconstructs with the best-predicted dosimetry as described hereinabovecan be assessed for their ability to serve as a vehicle for theradioimmunotherapy of solid tumors. Notably, diabodies with similaraffinities but contrasting impacts on in vitro growth (e.g., one diabodythat inhibits growth and one that does not) may be employed to determineif any growth inhibitory signaling is synergistic or additive withradioimmunotherapy. Inasmuch as scid mice have been found to behypersensitive to ionizing radiation due to defects in DNA repair(Biedermann, K. A., et al. (1991) Proc, Natl, Acad. Sci., 88:1394-1397),therapy trials can be performed in, for example, athymic nude mice.

Initially, the maximum tolerated dose (MTD) and LD₁₀ can be determinedas follows. The diabody will be labeled at a specific activityapproximating 5 mCi (180 MBq) of ¹³¹I per mg diabody. Notably, ⁹⁰Y and¹⁷⁷Lu, among other isotopes, can be used in place of ¹³¹I inradioimmunotherapy, particularly if the radioiodine-labeled diabodiesare rapidly internalized. A single i.v. dose, employing one of at leastabout five different doses, can be given to cohorts of about 5 non-tumorbearing athymic nude mice. The mice can then be observed for signs oftoxicity (e.g., weight loss, petechiae, and death) and the maximumtolerated dose can then be identified as that at which all of thetreated mice survive with less than about a 10% loss of body weight. TheLD₁₀ can be defined as the dose associated with 10% mortality. Inasmuchas the biodistribution data for diabodies indicate that the greatestdegree of normal organ retention occurs in the kidneys, renal retentionmay largely define the maximally-tolerated dose (MTD) in clinicalradioimmunotherpy trials. Accordingly, the long-term effects of thistherapy on the kidneys can also be determined. Briefly, the mice fromthese toxicity studies can be studied for about one year wherein monthlyblood samples can be acquired, for example, by retro-orbital bleeding toevaluate levels of markers that are associated with renal failure (e.g.,blood urea nitrogen and serum creatinine). After about one year, themice can be euthanized and their tissues can be fixed forhistopathological examination. Particular scrutiny can be placed uponthe evaluation of the kidneys and bone marrow.

Cohorts of approximately ten mice bearing subcutaneous OVCAR-3, IGROV-1,or SK-OV-3 tumors can be treated at the MTD for all schedules of eachmolecule to be tested. Duplicate experiments can be performed in micewith small (˜2 mm³) palpable tumors and a second set of treatmentstudies can be performed in mice bearing established (˜10 mm³) tumors.In all experiments, the radiolabeled diabody can be administered by i.v.bolus injection in a maximum volume of about 200 μL. The therapeuticeffects can be monitored by serially measuring tumor size (as describedhereinabove) and sacrificing animals whose tumors exceed about 2 cm ingreatest diameter and by plotting both tumor growth rates and times tosacrifice. All studies can include one or more of the following: groupsthat do not receive treatment, groups treated with radiolabeled growthinhibitory diabody, groups treated with radiolabeled non-growthinhibitory diabody with or without the same affinity, and groups treatedwith unlabeled forms of both diabodies.

As intraperitoneal (i.p.) models are particularly germane to ovariancancer, the above-described studies can be performed in nude mice at 7and 14 days following the i.p. injection of 4×10⁶ OVCAR-3, IGROV-1, orSK-OV-3 cells. In this model, evidence of abdominal distention typicallyoccurs after about 42 days and tumors are routinely observed within theperitoneum, within the intrabursal space and within the ovaries. Inthese studies, the mice can be followed for survival following themethods previously described in a study of antibody-based therapy ofovarian cancer in a SCID mouse model (Weiner, L. M., et al., (1993)Cancer Res., 53:94-100).

Future studies can examine the combined effect of anti-MISIIR diabodies(growth inhibitory and non-growth inhibitory) with subcurative doses ofchemotherapeutic agents that are active in ovarian cancer such as,without limitation, taxanes, platinum, carboplatin, and combinations oftwo or more agents such as gemcitabine with cisplatin, topotecan, and/ordoxorubicin. Additionally, the efficacy of anti-MISIIR diabody-basedradioimmunotherapy followed by sub-curative chemotherapy in the mousemodel can be examined.

The tumor targeting and dosimetry of anti-MISIIR antibodies in additionto toxicities and MTDs can also be determined in humans. For example,women with metastatic, measurable MISIIR expressing ovarian cancer, asin the human imaging trials described hereinabove, can receive a scoutdose consisting of an intravenous injection of 5 mCi (180 MBq) of ¹³¹Ion 5 mg of diabody. Whole body images can be acquired on a gamma cameraand blood samples can also be taken at about one hour, 4-8 hours, 24hours, 48 hours, 72 hours and 120 hours after administration. Regions ofinterest can be drawn around critical organs and the amount of localizedactivity can be determined. Blood samples can be counted along withstandards in a gamma well counter to determine the activity remaining incirculation. The projected radiation exposure to each organ and to bonemarrow associated with the patient's planned therapeutic dose can thenbe determined using the MIRD Dose 3 software package (Oak RidgeAssociated Universities). If the exposure is predicted to be within safelimits for all critical organs, the patient can be treated as describedbelow on day 8 using the optimal total diabody protein dose identifiedin the first trial described above. Cohorts of about 3-6 patients willbe treated in a standard dose-escalation schema. Dose escalation arepreferably not permitted until at least six weeks have elapsed followingtreatment of the last patient on a current dose cohort. An example ofdose escalation is seen in Table 1.

TABLE 1 Dose Radionuclide Dose Level Pts. Diabody Dose (mCi) 1 3  Fixed^(a) 10 mCi^(b) 2 3 Fixed 20 mCi 3 3 Fixed 40 mCi 4 3 Fixed 55mCi 5 3 Fixed 65 mCi 6 3-6 Fixed 75 mCi^(c) ^(a)The total diabodyprotein dose can be the optimal dose that was identified in the firsttrial. ^(b)If one dose-limiting toxicity (DLT) is observed in the firstthree patients at a given dose level, another three patients can beaccrued to that dose level. If >2/6 patients exhibit DLT in a given dosecohort, that dose can be considered to exceed the MTD, and an additional3-6 patients can be treated at the next highest dose level to confirm itdoes not exceed the MTD. ^(c)Dose-escalation in 5 mCi (180 MBq)increments can continue in three-patient cohorts until MTD has beenidentified. Six weeks must elapse from treatment of the last patient ina dose cohort before dose-escalation can occur.

Safety can be determined by the ongoing monitoring of the incidence andintensity of adverse events graded by, for example, the Common ToxicityCriteria (see, e.g., www.fda.gov/cder/cancer). Following injection ofthe radiopharmaceutical scout dose, whole body gamma camera imaging,optionally with SPECT (single photon emission computed tomography)capabilities, can be performed immediately after administration at thetime points indicated above. The sensitivity and specificity of gammacamera imaging can be determined by comparing SPECT images with CTscans. Therapeutic efficacy can be determined by comparing pre- andpost-treatment CT scans of known tumor deposits (CT scans can beacquired every six weeks for the first three months following therapyand then every two months thereafter until there is evidence ofprogression). As the diabody is a novel construct, regular blood samplescan be obtained regularly to assay for the development of humananti-diabody antibodies (HADA). Statistical analysis of the results canbe performed as described hereinabove in Example 5. As an example, thetherapeutically effective dosage amount for a patient may be 50-200 mCi.

While the above demonstrates the therapeutic use of anti-MISIIR antibodymolecules conjugated to radioisotopes, similar studies can be performedfor immunotoxins and anti-MISIIR antibody molecules conjugated tochemotherapeutic agents.

While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

1. An isolated antibody having binding affinity for Mullerian InhibitingSubstance Type II Receptor (MISIIR), wherein said antibody is selectedfrom the group consisting of IgG, Fab, Fab′, Fv, and F(ab′)₂, andwherein the amino acid sequences of the V_(H) and V_(L) domains of saidantibody correspond to the V_(H) and V_(L) domains of one of SEQ ID NO:9-15. 2-11. (canceled)
 12. The antibody of claim 1, wherein saidantibody is conjugated to a radioisotope.
 13. The antibody of claim 12,wherein said radioisotope is selected from the group consisting of ⁹⁰Y,¹⁷⁷Lu, ¹³¹I, and ¹⁸⁶Re.
 14. A composition comprising an antibody ofclaim 1 and a pharmaceutically acceptable carrier.
 15. The antibody ofclaim 1, wherein said antibody is conjugated to at least one moleculeselected from the group consisting of a radioisotope, a detectablelabel, a toxin, a magnetic bead, a pro-drug, a pro-drug convertingenzyme, and a chemotherapeutic agent. 16-18. (canceled)
 19. The antibodyas claimed in claim 15, wherein said molecule is a toxin selected fromthe group consisting of saprin, ricin, abrin, ethidium bromide,diptheria toxin, Pseudomonas exotoxin, PE40, PE38, saporin, gelonin,RNAse, protein nucleic acids (PNAs), ribosome inactivating protein(RIP), type-1 or type-2, pokeweed anti-viral protein (PAP), bryodin,momordin, and bouganin.
 20. A method for treating cancer, comprisingadministering to a patient in need thereof a therapeutically effectiveamount of the composition of claim
 14. 21. (canceled)
 22. The method ofclaim 20, wherein said cancer is selected from the group consisting ofbreast, prostate, cervical, ovarian, testicular, and pulmonary cancers.23. (canceled)
 24. The method of claim 20, further comprisingadministration of at least one chemotherapeutic agent. 25-30. (canceled)31. A method for imaging cancer in a patient comprising administering toa patient an antibody of claim 1, said antibody optionally comprising adetectable label.
 32. (canceled)
 33. The method of claim 31, whereinsaid detectable label comprises a radioisotope.
 34. The method of claim31, wherein said cancer is selected from the group consisting of breast,prostate, cervical, ovarian, testicular, and pulmonary cancers.
 35. Themethod of claim 34, wherein said cancer is ovarian cancer.
 36. Themethod of claim 33, wherein said radioisotope is ¹²⁴I.
 37. A method fordetecting the presence or absence of MISIIR in a biological samplecomprising: a) providing a biological sample; b) incubating said samplein the presence and absence of the antibody of claim 1, said antibodybeing detectably labeled; c) determining the presence or absence of saidMISIIR as a function of the amount of detectable label bound to saidsample.
 38. The method of claim 37, further comprising the use of anon-specific antibody as a negative control.
 39. The method of claim 37,wherein said biological sample is selected from the group consisting ofblood, plasma, urine, tumor cells, blood cells, ovarian cells, prostatecells, breast cells, testicular cells, cervical cells, lung cells, andnipple aspirates. 40-42. (canceled)
 43. The antibody of claim 1, whereinthe amino acid sequences of the V_(H) and V_(L) domains of said antibodycorrespond to the V_(H) and V_(L) domains of SEQ ID NO:
 13. 44. Theantibody of claim 1, wherein said antibody is an IgG.